Internalization and trafficking of the human and rat growth hormone-releasing hormone receptor

Internalization and intracellular trafficking of the growth hormone-releasing hormone receptor (GHRH-R) were studied in rat anterior pituitary and human (h) and rat(r) GHRH-R-transfected BHKcells, with the GHRH agonist, [N alpha-5-carboxyfluoresceinyl-D-Ala(2), Ala(8), Ala(15), Lys(22)]hGHRH(1-29)NH2 (Fluo-GHRH). Time- and temperature-dependent internalization of stimulated GH RH-R was blocked by phenyl arsi ne oxide (PAO) in both cell types. In anterior pituitary and rGHRH-R-transfected BHK cells, only filipin III and cerulenin blocked receptor-mediated internalization of Fluo-GHRH while in hGHRH-R-tmnsfected BHK cells, only hyperosmolar sucrose inhibited this process. These results suggest that hGHRH-R internalization is clathrin-dependent, while fatty acid acylation of rGHRH-R appears to be a prerequisite to caveolin-clependent internalization. Experiments in anterior pituitary using Bodipy-FL-C5 ganglioside GM1, a specific marker of lipid rafts such as caveolae, confirmed this latter pathway. Colocalization of Fluo-GHRH with LysoTracker indicated that Fluo-GHRH was directed to acidic organelles in both cell types. Finally, studies using cycloheximide and monensin showed that upon stimulation with GHRH, an optimal concentration of functional GHRH-Rwas maintained at the plasma membrane due to de novo synthesis and recycling in pituitary cells and to de novo synthesis solely in hGHRH-R-transfected BHK cells. This first study on the dynamics of the GHRH/GHRH-R complexes using fluorescence imaging in a native environment compared to cell system models, revealed that both receptor primary structure and concentration at the plasma membrane play important roles in internalization and trafficking of specific G-protein-coupled receptors (GPCR). J. Cell. Physiol. 203: 335-344, 2005. (c) 2004 Wiley-Liss, Inc.